The periodontopathic microorganism Porphyromonas gingivalis expresses a number of potential virulence factors including cysteine proteases and hemagglutinins. However, experimental data documenting these factors as true virulence factors is lacking. The recent isolation of genes coding for several of these factors and their utilization in constructing specific P. gingivalis mutants which can be introduced into animal models offers an optimal approach for defining virulence factors in these organisms. The present proposal will examine the contribution of two cysteine proteases, Arg-gingipain- 1 and PrtT, to the virulence of P. gingivalis. Using molecular genetic approaches, the environmental factors regulating the expression of these enzymes will be examined. Mutants constructed from the genes for each enzyme will also be utilized in a rat bone loss model system in order to assess their relative contributions to the virulence of these organisms. In addition, the molecular basis for the expression of hemagglutinating activity by these enzymes will be explored. This proposal will also specifically examine the hypothesis that several of the cysteine proteases are composed of two functional domains: one involved in proteolysis and the other acting as an adhesin. Since P gingivalis can utilize hemin derived from hemoglobin as an iron source in the environment of the inflamed periodontal pocket, the mechanisms of hemin uptake by these organisms will be investigated. This property is likely an important regulator of bacterial colonization of the gingival margin. Therefore, the hemR gene recently identified in this laboratory as a candidate hemin receptor will be further characterized. In addition, the other genes present downstream from hemR in the hemin regulated operon will be isolated and characterized. It will also be of interest to determine how environmental iron levels regulate the expression of this operon. The results derived from the present proposal will help to define virulence factors of P. gingivalis for future targeting in anti- periodontitis strategies.